Molecular characterisation and functional analysis of eEF1B subunits in mammals
Botelho Duarte Portela, Miriam
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During the elongation of the polypeptide chain in eukaryotic protein synthesis, GTP-bound eukaryotic translation elongation factor 1A recruits the aminoacyl tRNA to the A-site of the ribosome. The GDP-GTP recycling is catalysed by the elongation factor 1B complex (eEF1B) which in higher eukaryotes consists of three different subunits: alpha, delta and gamma. Previous studies on eEF1B focused mainly on biochemical analysis and reports of overexpression in tumours and correlation to decreased survival rate but not a lot is known about is biology. The aim of this PhD is to characterise the eEF1B subunits at the molecular level in view of their potential involvement in tumourigenesis using a variety of bioinformatic and laboratory techniques. All three subunits were found to be ubiquitously expressed at mRNA and protein levels in all mouse tissues analysed. In addition, eEF1Bβ has several transcript variants in mice derived from alternative splicing and multiple isoforms, including a brain and testis specific heavier isoform and a muscle-specific form in addition to other forms. The characteristics of each eEF1B subunit were catalogued by further bioinformatic analysis. eEF1Bα was not detectable at early mouse developmental stages, eEF1Bβ showed stronger expression at pre-natal and early post-natal stages than adult stage whereas eEF1Bγ is ubiquitously expressed at similar levels throughout mouse development. In adult mice and human tissues, eEF1B subunits appeared to be expressed in different cell types and cell sub-populations. Surprisingly, cytoplasmic and some nuclear expression was observed in vivo. This nuclear expression pattern could not be observed in cell lines and it was not related to the cell cycle stage in vitro. The expression of eEF1B subunits did not change during the cell cycle except eEF1Bγ which was highly expressed in S-phase arrested cells. Knockdown by siRNAs of eEF1B subunits leads to decreased proliferation, increased number of cells in G0/G1 phase and increase in apoptosis in HeLa, HCT116, DLD1 and HepG2 cells. In contrast, overexpression in HeLa cells with a V5-tagged constructs lead to increased proliferation, increased number of cells in the G2/M phase and increased viability. Knockdown of eEF1Bα and eEF1Bβ leads to a reduction in eEF1Bγ levels; it is therefore possible that the phenotype shown by the knockdown of each subunit individually might be due to the reduced levels of eEF1Bγ. However, overexpression of each subunit did not affect the protein levels of the other subunits. The presence of multiple forms, the complex expression pattern and distribution of each eEF1B subunit in mouse and human tissues, and the knockdown and overexpression effect on cells suggests that the eEF1B complex might have different quaternary forms throughout development and in different cell types, possibly a more intricate role in translation, potential non-canonical functions any of which may be implicated in the potential role of eEF1B subunits in tumourgenesis.